The goal of this project is to identify transcriptional mechanisms underlying Sox9 expression in chondrocytes. Sox9 is a master chondrogenic transcription factor and thus an attractive target for urgently needed therapies for chondrodysplasias and joint degeneration diseases. Since Sox9 is primarily controlled at the transcriptional level, the best way to stimulate its activity in these diseases is to use its natural transcriptional mechanisms. These mechanisms remain elusive, but we identify in preliminary studies two highly conserved sequences far upstream of Sox9 that are potent chondrocyte-specific enhancers. These enhancers have overlapping domains of activity in all cartilage structures in vivo and synergize with each other in vitro. The core region of the first enhancer features putative sites for multiple types of transcription factors, but the specific factors, referred to as chondrogenin, which mediate its activity are unknown. The other enhancer is upregulated by Sox9 itself in vitro, as is the endogenous Sox9 gene, and it features multiple Sox-binding sites. These data suggest that chondrogenin activates Sox9 expression by binding to the first enhancer and that Sox9 binds to the other enhancer to synergize with chondrogenin and to maintain its own gene expression. We propose four specific aims to test this hypothesis. Aim 1 is to use a transgenic mouse approach to determine whether the two enhancers may be sufficient to activate the Sox9 promoter in cartilage throughout life. Aim 2 is to use conditional inactivation approaches to test whether the enhancers are necessary to activate the endogenous Sox9 gene in cartilage. Aim 3 is to use complementary genetic, cellular and molecular approaches to demonstrate that Sox9 binds to the second enhancer in chondrocytes, does so through specific sites, and thereby upregulates its own gene expression. Aim 4 is to use candidate and biochemical approaches to identify the chondrogenin factors that mediate the activity of the first enhancer and to determine their roles in chondrogenesis. We anticipate that the results of this work will have high scientific and technological impact. We expect to identify Sox9 enhancers and upstream transcription factors that are essential for chondrogenesis and that will therefore become relevant targets to upregulate Sox9 expression in new treatments for cartilage malformation and degeneration diseases.